Project Summary Adjuvant options for subunit vaccine design are extremely limited. Apart from alum, just two adjuvants are approved for use in the US: squalene and the combination adjuvant system AS04, which combines traditional alum with a partial agonist of TLR4, monophosphoryl lipid A (MPL). MPL adjuvant is notable because it is the first and only purified TLR agonist in a licensed vaccine in the US, was tested extensively for safety and efficacy in large clinical trials, and shows promise as an adjuvant in ambitious vaccine projects such as the RTS,S/AS01 vaccine to prevent malaria. Alum+MPLA is currently the best example we have of a clinically successful adjuvant but the factors that make it successful are poorly understood. Moreover, MPL is a heterogeneous mixture of lipid A structures in which a hexa-acylated structure is thought to be the ?active? component, but the functions of other components have never been tested in human immune cells. Preliminary studies support the hypothesis that MPL adjuvant contains an immunomodulatory inhibitor of human TLR4 that constrains endotoxic responses with less impact on adaptive priming. This project will identify the inhibitor, determine if and how it has differential effects on MyD88- vs TRIF-signaling, and evaluate synthetic analogues of MPL with amplified properties as adjuvants.